mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells
Authors: Sanne Bevers, Sander A.A. Kooijmans, Elien Van de Velde, Martijn J.W. Evers, Sofie Seghers, Jerney J.J.M. Gitz-Francois, Nicky C.H. van Kronenburg, Marcel H.A.M. Fens, Enrico Mastrobattista, Lucie Hassler, Helena Sork, Taavi Lehto, Kariem E. Ahmed, Samir El Andaloussi, Katja Fiedler, Karine Breckpot, Michael Maes, Diane Van Hoorick, Thierry Bastogne, Raymond M. Schiffelers, Stefaan De Koker
Journal: Molecular Therapy (2022)

mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy. mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy. IntroductionMessenger RNA (mRNA) vaccines are extremely versatile, as mRNA sequences can be easily tailored to encode any antigen of interest, enabling both rapid and mass-scale vaccine development against emerging pathogens as well as personalized vaccine design against cancers. Key to the success of the currently approved SARS-CoV2 mRNA vaccines and of many mRNA-based prophylactic vaccines in clinical development are lipid-based nanoparticles (LNPs), delivery vehicles that mediate efficient mRNA expression in situ and endow the vaccine with intrinsic adjuvant properties.1Alameh M.-G.G. Tombácz I. Bettini E. Lederer K. Sittplangkoon C. Wilmore J.R. Gaudette B.T. Soliman O.Y. Pine M. Hicks P. et al.Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.Immunity. 2021; 54: 2877-2892.e7Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar LNPs are composed of an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid, with the ionizable lipid being considered the most important driver of mRNA expression. In contrast to their predecessors having a cationic charge across a wide variety of pH ranges, ionizable lipids are pH-sensitive lipids that endow the LNPs with a positive, membrane fusion-promoting charge at endosomal pH while having a neutral charge at physiological pH.2Reichmuth A.M. Oberli M.A. Jeklenec A. Langer R. Blankschtein D. mRNA vaccine delivery using lipid nanoparticles.Ther. Deliv. 2016; 7: 319-334Crossref PubMed Scopus (263) Google Scholar The ionizable lipid is required to encapsulate the mRNA and drives endosomal escape. The PEGylated lipid improves LNP stability and controls the interaction of the LNPs with blood proteins and cells. Cholesterol and the phospholipid contribute to LNP stability and endosomal membrane destabilization.2Reichmuth A.M. Oberli M.A. Jeklenec A. Langer R. Blankschtein D. mRNA vaccine delivery using lipid nanoparticles.Ther. Deliv. 2016; 7: 319-334Crossref PubMed Scopus (263) Google Scholar Intramuscular3Cafri G. Gartner J.J. Zaks T. Hopson K. Levin N. Paria B.C. Parkhurst M.R. Yossef R. Lowery F.J. Jafferji M.S. et al.mRNA vaccine–induced neoantigen-specific T cell immunity in patients with gastrointestinal cancer.J. Clin. Invest. 2020; 130: 5976-5988Crossref PubMed Scopus (85) Google Scholar or subcutaneous4Oberli M.A. Reichmuth A.M. Dorkin J.R. Mitchell M.J. Fenton O.S. Jaklenec A. Anderson D.G. Langer R. Blankschtein D. Lipid nanoparticle assisted mRNA delivery for potent cancer immunotherapy.Nano Lett. 2017; 17: 1326-1335Crossref PubMed Scopus (331) Google Scholar administration of mRNA-LNPs has been reported to elicit CD8 T cell responses against the mRNA encoded antigen. In these strategies, mRNA is delivered to local antigen-presenting cells (APCs), resulting in low to moderate antigen-specific CD8 T cell levels in the circulation. However, to effectively combat tumors (especially at distant locations) potent, long-lasting, and systemic CD8 T cell responses are required. Intravenous (i.v.) delivery of mRNA vaccines has been described to confer enhanced T cell responses and antitumor immunity,5Kranz L.M. Diken M. Haas H. Kreiter S. Loquai C. Reuter K.C. Meng M. Fritz D. Vascotto F. Hefesha H. et al.Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy.Nature. 2016; 534: 396-401Crossref PubMed Scopus (854) Google Scholar, 6Broos K. Van der Jeught K. Puttemans J. Goyvaerts C. Heirman C. Dewitte H. Verbeke R. Lentacker I. Thielemans K. Breckpot K. Particle-mediated intravenous delivery of antigen mRNA results in strong antigen-specific T-cell responses despite the induction of type I interferon.Mol. Ther. Nucleic Acids. 2016; 5: e326Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 7Van Der Jeught K. De Koker S. Bialkowski L. Heirman C. Tjok Joe P. Perche F. Maenhout S. Bevers S. Broos K. Deswarte K. et al.Dendritic cell targeting mRNA lipopolyplexes combine strong antitumor T - cell immunity with improved inflammatory safety.ACS Nano. 2018; 12: 9815-9829Crossref PubMed Scopus (57) Google Scholar which is likely related to its capacity to mobilize the large pools of APCs present in the spleen. Induction of strong CD8 T cell responses has been confirmed clinically, upon i.v. immunization of metastatic melanoma patients with mRNA lipoplexes composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and the cationic lipid DOTMA,8Sahin U. Oehm P. Derhovanessian E. Jabulowsky R.A. Vormehr M. Gold M. Maurus D. Schwarck-Kokarakis D. Kuhn A.N. Omokoko T. et al.An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma.Nature. 2020; 585: 107-112Crossref PubMed Scopus (231) Google Scholar but no LNP-based formulation has yet been used in the clinic for i.v. therapeutic cancer vaccination. LNPs might show even further improved activity compared with lipoplexes given their enhanced intrinsic potential to transfect cells. Nonetheless, standard LNP formulations typically deliver RNA to hepatocytes upon i.v. delivery,9Kauffman K.J. Dorkin J.R. Yang J.H. Heartlein M.W. Derosa F. Mir F.F. Fenton O.S. Anderson D.G. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs.Nano Lett. 2015; 15: 7300-7306Crossref PubMed Scopus (295) Google Scholar, 10Ramaswamy S. Tonnu N. Tachikawa K. Limphong P. Vega J.B. Karmali P.P. Chivukula P. Verma I.M. Systemic delivery of factor IX messenger RNA for protein replacement therapy.Proc. Natl. Acad. Sci. USA. 2017; 114: E1941-E1950Crossref PubMed Scopus (159) Google Scholar, 11Pardi N. Tuyishime S. Muramatsu H. Kariko K. Mui B.L. Tam Y.K. Madden T.D. Hope M.J. Weissman D. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes.J. Control. Release. 2015; 217: 345-351Crossref PubMed Scopus (366) Google Scholar instead of the APCs needed to instigate CD8 T cells. In this study, we speculated that LNPs could be tailored toward systemic vaccination through optimization of the molar ratio between the four lipids and selection of an appropriate polyethylene glycol (PEG) lipid. These parameters are known to impact LNP features such as size, ζ potential, pharmacokinetics, and biodistribution, which ultimately will determine in vivo immunogenicity.4Oberli M.A. Reichmuth A.M. Dorkin J.R. Mitchell M.J. Fenton O.S. Jaklenec A. Anderson D.G. Langer R. Blankschtein D. Lipid nanoparticle assisted mRNA delivery for potent cancer immunotherapy.Nano Lett. 2017; 17: 1326-1335Crossref PubMed Scopus (331) Google Scholar,9Kauffman K.J. Dorkin J.R. Yang J.H. Heartlein M.W. Derosa F. Mir F.F. Fenton O.S. Anderson D.G. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs.Nano Lett. 2015; 15: 7300-7306Crossref PubMed Scopus (295) Google Scholar,12Chen S. Tam Y.Y.C. Lin P.J.C. Sung M.M.H. Tam Y.K. Cullis P.R. Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA.J. Control. Release. 2016; 235: 236-244Crossref PubMed Scopus (129) Google Scholar, 13Chen D. Ganesh S. Wang W. Amiji M. The role of surface chemistry in serum protein corona-mediated cellular delivery and gene silencing with lipid nanoparticles.Nanoscale. 2019; 11: 8760-8775Crossref PubMed Google Scholar, 14Suzuki T. Suzuki Y. Hihara T. Kubara K. Kondo K. Hyodo K. Yamazaki K. Ishida T. Ishihara H. PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: faster PEG shedding attenuates anti-PEG IgM production.Int. J. Pharm. 2020; 588: 119792Crossref PubMed Scopus (25) Google Scholar, 15Hassett K.J. Benenato K.E. Jacquinet E. Lee A. Woods A. Yuzhakov O. Himansu S. Deterling J. Geilich B.M. Ketova T. et al.Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines.Mol. Ther. Nucleic Acids. 2019; 15: 1-11Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 16Hassett K.J. Higgins J. Woods A. Levy B. Xia Y. Hsiao C.J. Acosta E. Almarsson Ö. Moore M.J. Brito L.A. Impact of lipid nanoparticle size on mRNA vaccine immunogenicity.J. Control. Release. 2021; 335: 237-246Crossref PubMed Scopus (41) Google Scholar To design and screen a virtually unlimited variety of potential LNP compositions in a time- and cost-effective manner, we adopted a quality-by-design approach based on a statistical design-of-experiment (DOE) coupled to Bayesian regression modeling. This strategy enabled us to identify the key composition parameters that determine immunogenicity and to predict optimal LNP compositions within a single round of in vivo immunogenicity assessment. Optimal LNP compositions (within the constraints of the design space) resulted in strong CD8 T cell responses that could be boosted by repeated administration and conveyed antitumor efficacy in a syngeneic mouse TC-1 tumor model. Mechanistic studies revealed that the T cell response was dependent on initial mRNA expression by various APCs in the spleen and on the induction of type I interferons (IFNs). Surprisingly, we also discovered a previously unrecognized role of B cells in instigating antigen-specific CD8 T cell responses upon intravenous mRNA-LNP vaccination.ResultsDOE-driven optimization of LNP composition maximizes T cell responsesWe aimed to maximize the T cell response elicited by antigen-encoding mRNA-LNPs composed of the ionizable lipid Coatsome SS-EC,17Akita H. Noguchi Y. Hatakeyama H. Sato Y. Tange K. Nakai Y. Harashima H. Molecular tuning of a vitamin E-scaffold pH-sensitive and reductive cleavable lipid-like material for accelerated in vivo hepatic siRNA delivery.ACS Biomater. Sci. Eng. 2015; 1: 834-844Crossref PubMed Scopus (35) Google Scholar cholesterol, DOPE, and a PEGylated lipid. The human papillomavirus 16 (HPV16) oncoprotein E7 was used as the antigen of choice. HPV16 is a frequent cause of cervical, anal, and oropharyngeal cancer. The oncogenic potential of HPV is driven by persistent expression of the viral oncoproteins E6 and E7, which cause degradation of the tumor-suppressor proteins p53 and Rb.18Pal A. Kundu R. Human Papillomavirus E6 and E7: the cervical cancer hallmarks and targets for therapy.Front. Microbiol. 2020; 10: 3116Crossref PubMed Scopus (122) Google Scholar Whereas E6 is not immunogenic in standard inbred mice strains,19Grunwitz C. Salomon N. Vascotto F. Selmi A. Bukur T. Diken M. Kreiter S. Türeci Ö. Sahin U. Grunwitz C. et al.HPV16 RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positive mouse tumors and establishes protective T cell memory.Oncoimmunology. 2019; 8: e1629259Crossref PubMed Scopus (29) Google Scholar E7 contains a well-established class I epitope recognized in C57BL/6 mice, making it a highly relevant and suitable antigen to assess the potency of vaccine platforms in mice. Using DOE methodology, an LNP library was designed to address the impact of lipid molar ratio and PEG-lipid chemistry on the T cell response. Three PEGylated lipids (DMG-PEG2000, DSG-PEG2000, and DSPE-PEG1000) that differ in length and chemistry of the acyl chain and/or the molecular weight of PEG were explored. Per PEG-lipid chemistry, LNPs were formulated at 11 different molar ratios of the four lipid components, yielding a library of 33 LNPs (Table S1). These 11 molar ratios were derived from a Roquemore hybrid design20Lewis G.A. Mathieu D. Phan-Tan-Lu R. Pharmaceutical Experimental Design. Marcel Dekker, Inc, 1999Google Scholar in which the molar percentage of ionizable lipid, DOPE, and PEG lipid each had five levels. Cholesterol was used as a “filler” variable that completed the total molar percentage to 100% (Figure 1A ). This design is particularly suited for the generation of quadratic response surface models and more economic than traditional central composite or Box-Behnken designs (15 and 13 conditions required, respectively). The composition and properties (size, polydispersity index, percentage of mRNA, encapsulation, and ζ potential) of the LNPs are shown in Table S1. All LNPs remained stable at 4°C for 1 month (Figure S1). LNPs were administered i.v. three times at weekly intervals. Five days after the third injection, the percentage of E7-specific CD8 T cells in blood was determined. Data were subjected to Bayesian statistical modeling to identify the LNP components critical for activity (Figure 1B). Results showed that the magnitude of the CD8 T cell response heavily depends on the LNP composition (Figure 1C). Bayesian effect analysis (supplemental methods) identified PEG-lipid chemistry and molar percentage of PEG lipid as the most critical LNP attributes. In the cases of DSG-PEG2000- and DSPE-PEG1000-based LNPs, the percentage of ionizable lipid also had a critical impact. Interaction effects were found between the percentages of DOPE and the PEG lipids, indicating that LNP optimization required fine-tuning of all lipid components simultaneously. Bayesian regression modeling was applied to predict the immunogenicity of LNP compositions not included in our initial library (Figure 1D). For all PEG-lipid types, the predicted optimal T cell response region corresponded to high percentages of the ionizable lipid SS-EC (>50%) and low percentages of PEG lipid (<0.6%) and DOPE (<10%). To validate the predictive value of the models, we tested six new LNP compositions (LNP34–LNP39, Table S1) in vivo. T cell responses higher than or equal to the best performers of the original library were predicted for mice immunized with LNP34 (DMG-PEG2000), LNP36 (DSG-PEG2000), and LNP38 (DSPE-PEG1000) (optimal LNPs), whereas poor T cell responses were predicted for LNP35 (DMG-PEG2000), LNP37 (DSG-PEG2000), and LNP39 (DSPE-PEG1000) (non-optimal LNPs). The experimental data largely matched the predictions and validated the models in the cases of DMG-PEG2000 and DSG-PEG2000 LNPs (Figures 1E and 1G; supplemental methods). In contrast to the predictions, LNP39 elicited T cell responses nearly equal to those of LNP38 (Figure 1F), meaning that the DSPE-PEG1000 LNP model could not be validated. Because of this unpredictable behavior and visual toxicity in mice (lethargy, pain symptoms) DSPE-PEG1000 LNPs were excluded from further assessment.mRNA-LNP vaccines induce qualitative T cell responses and tumor regressionThe success of cancer immunotherapy is determined by a multitude of factors, including the T cell phenotype and functionality. We first assessed the quality of the T cell response evoked by the optimal LNPs (LNP34 and LNP36). Therefore, mice received three weekly immunizations followed by a final immunization at day 50. E7 mRNA was supplemented with TriMix, which a mix of three mRNAs encoding murine CD70, murine CD40L, and a constitutively active TLR4, which increases the strength of the T cell response.21Bonehill A. Tuyaerts S. Van Nuffel A.M. Heirman C. Bos T.J. Fostier K. Neyns B. Thielemans K. Enhancing the T-cell stimulatory capacity of human dendritic cells by co-electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA.Mol. Ther. 2008; 16: 1170-1180Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Expression of all four co-formulated mRNAs was confirmed by in vitro transfection (data not shown). For both LNPs, the kinetics and phenotypic traits of the T cell response were similar. Following three immunizations with E7-TriMix LNPs, over 70% of CD8 T cells in blood were E7 specific (Figure 2A ). Five weeks after the third immunization (day 49), the percentage of E7-specific CD8 T cells remained highly elevated (approximately 40%, Figure 2A) with the majority displaying a short-lived effector cell phenotype (KLRG1+ CD127−) (Figures 2B and 2C). However, a noteworthy population (∼10%) of memory precursor effector cells (KLRG1− CD127+) was present as well. Functional memory conversion was evidenced by the rapid re-expansion of E7-specific CD8 T cells after the day-50 booster immunization (Figures 2A and 2C). IFN-γ concentrations in serum increased with every immunization (Figure 2D), indicating increased systemic immune stimulation. To assess T cell functionality, we performed an intracellular cytokine staining on splenocytes obtained from mice after three immunizations. Polyfunctional CD8 T cells, which produce more than one cytokine simultaneously, are associated with better control of infectious diseases22Almeida J.R. Price D.A. Papagno L. Arkoub Z.A. Sauce D. Bornstein E. Asher T.E. Samri A. Schnuriger A. Theodorou I. et al.Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover.J. Exp. Med. 2007; 204: 2473-2485Crossref PubMed Scopus (573) Google Scholar,23Ciuffreda D. Comte D. Cavassini M. Giostra E. Bühler L. Perruchoud M. Heim M.H. Battegay M. Genné D. Mulhaupt B. et al.Polyfunctional HCV-specific T-cell responses are associated with effective control of HCV replication.Eur. J. Immunol. 2008; 38: 2665-2677Crossref PubMed Scopus (115) Google Scholar and tumors24Wimmers F. Aarntzen E.H.J.G. Duiveman-deBoer T. Figdor C.G. Jacobs J.F.M. Tel J. de Vries I.J.M. Long-lasting multifunctional CD8+ T cell responses in end-stage melanoma patients can be induced by dendritic cell vaccination.Oncoimmunology. 2016; 5: e1067745Crossref PubMed Scopus (40) Google Scholar, 25Spranger S. Koblish H.K. Horton B. Scherle P.A. Newton R. Gajewski T.F. Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8+ T cells directly within the tumor microenvironment.J. Immunother. Cancer. 2014; 2: 3Crossref PubMed Scopus (407) Google Scholar, 26De Groot R. Van Loenen M.M. Guislain A. Nicolet B.P. Freen-Van Heeren J.J. Verhagen O.J.H.M. Van Den Heuvel M.M. De Jong J. Burger P. Van Der Schoot C.E. et al.Polyfunctional tumor-reactive T cells are effectively expanded from non-small cell lung cancers, and correlate with an immune-engaged T cell profile.Oncoimmunology. 2019; 8: e1648170Crossref PubMed Scopus (24) Google Scholar and accounted for approximately 11% of CD8 T cells for both optimal LNPs (Figures 2E and 2F). To assess whether the activity of the LNPs also extended to other antigens, mRNA encoding the MC38 colon carcinoma-derived neo-epitope Adpgk or the B16-F10-derived melanocyte differentiation antigen Trp-2 was mixed with TriMix and encapsulated in LNP36. Augmented T cell responses were observed after two immunizations with LNPs containing both types of antigen-encoding mRNAs, especially when combined with TriMix (Figure S2). Next, therapeutic antitumor efficacy was assessed in mice bearing subcutaneous TC-1 tumors, which express HPV16 E7 antigen.27Lin K.Y. Guarnieri F.G. Staveley-O’Carroll K.F. Levitsky H.I. August J.T. Pardoll D.M. Wu T.C. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen.Cancer Res. 1996; 56: 21-26PubMed Google Scholar Treatment was initiated when tumors reached a mean diameter of 52 ± 4 mm3 (Figure 2G). E7-TriMix treatment resulted in profound tumor growth inhibition of the established TC-1 tumors (Figure 2H) and significantly prolonged survival time (Figure 2I), yet tumors relapsed after cessation of treatment. We additionally assessed the capacity of mRNA-LNP-elicited T cells to reach the tumor bed. Two i.v. injections with the mRNA-LNPs led to a strong infiltration of CD8+ tumor-infiltrating lymphocytes (TILs) into the tumor (Figures 2J and 2K), with over 70% being specific for E7 (Figure 2L). Treatment with LNP36 delivering TriMix without E7 or containing an irrelevant mRNA (Firefly luciferase [Fluc]) did not induce regression of tumors, nor did it result in increased percentages of E7-specific CD8 T cells in the tumor (Figures S3A and S3B). To understand why tumors relapse despite the high numbers of vaccine-elicited E7-specific CD8 T cells, we compared tumor E7 expression levels in LNP36 versus Tris-buffered saline (TBS)-treated tumors. Tumors that relapsed after vaccination with E7-TriMix delivered by LNP36 showed a profound decrease in E7 expression levels compared with TBS (Figure S3C), suggesting loss of antigen expression as a major mechanism of immune escape. Moreover, at the time of relapse (day 35) tumors were less infiltrated by E7-specific CD8 TILs compared with 2 days after the second immunization (day 17, regression phase) (Figure S3B). In addition, TILs in regressing tumors showed proliferative potential (Ki67+) and cytolytic capacities (granzyme B+), whereas TILs present in relapsing tumors predominantly had a non-proliferative (Ki67−), non-cytolytic (granzyme B−) phenotype and expressed high levels of the immune-checkpoint PD-1 (Figures S3D and S3E). This “non-reactive” phenotype might be inflicted by immune-suppressive factors present in the tumor microenvironment or driven by the lack of E7 presence. Further immunophenotyping revealed no significant increases in the levels of myeloid-derived suppressor cells, regulatory T cells, or M2 macrophages in relapsing tumors compared with regressing tumors (Figure S3F), arguing against a major role of these cells in the observed tumor escape.Figure 2Optimal mRNA-LNPs induce qualitative T cell responses and strong antitumor efficacyShow full caption(A) Mice were immunized with optimal LNPs encapsulating 5 μg of E7 and 5 μg of TriMix mRNA at days 0, 7, 14, and 50. Blood CD8 E7-specific T cell responses were determined at days 5, 20, 49, and 55. (B and C) Differential expression of KLRG1 and CD127 on blood E7-specific (+) and non-specific (−) CD8 T cells at day 49 and day 55. Representative contour plots (B, day 49) and quantification (C, mean ± SD [n = 4]) of KLRG1 and CD127 expression are shown. (D) IFN-γ levels in serum were measured 6 h after each immunization (n = 4–6). (E and F) IFN-γ and TNF-α expression by splenic CD8 T cells after three immunizations with TBS or mRNA-LNPs (weekly intervals, 10 μg of E7-TriMix). Representative contour plots (E) and quantification (F: mean ± SD [n = 3 for TBS, n = 5 for LNPs]) is shown. (G) When TC-1 tumors reached a mean volume of 52 mm3, mice were injected i.v. with 5 μg of E7-TriMix mRNA-LNPs (weekly intervals). (H) TC-1 growth curves in mice. Data are shown as mean ± SEM (n = 6–7). (I) Kaplan-Meier survival curves of TC-1-bearing mice. (J and K) Representative contour plots (J) and graph (K, mean ± SD [n = 4] with individual data points shown in red) quantifying the infiltration of CD8 T cells in TC-1 tumors. (L) E7 specificity of CD8 TILs in TC-1 tumors. Data are shown as mean ± SD with individual data points in red (n = 4). Statistical differences were assessed using a Mantel-Cox log-rank test with Bonferroni multiple testing correction (I), one-way ANOVA with Tukey post hoc test (J), or two-way Student’s t test (L).∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Early LNP association with splenic APCs correlates with immunogenicityTo address whether the magnitude of the T cell responses after the third immunization can be correlated to the initial biodistribution of mRNA uptake and expression at organ level and cell-type level, we encapsulated a mixture of Cy5-labeled (25% substituted cyanine-5 uridine-5′-triphosphate [Cy5-UTP], 75% substituted 5-methoxy UTP), and unlabeled (and without nucleoside modification) Fluc mRNA in the LNPs screened in the original immunogenicity assessment (Figure 3A ). Fluc activity was measured in liver, spleen, lung, heart, and kidney homogenates 4 h after LNP injection. LNP composition had a strong impact on the intensity and organ specificity of mRNA expression (Figure 3B). Across the library, Fluc expression was highest in the liver, followed by the spleen (Figure 3C), but the liver-to-spleen ratio differed strongly between LNPs. The magnitude of the E7-specific CD8 T cell responses positively correlated with absolute (DSG-PEG2000 LNPs) and relative (DMG-PEG2000 LNPs) spleen expression (Table S2). The importance of organ-specific mRNA delivery was further highlighted by the absence of correlation between total expression and T cell responses (Table S2). Significant correlations were also identified between LNP size (in turn strongly dependent on lipid composition) and relative expression in the spleen (Figure S4A) and between LNP size and the E7-specific CD8 T cell response (Figure S4B). Using Cy5-labeled mRNA as a fluorescent reporter molecule, we further assessed whether T cell responses can be linked to early (4 h post administration) mRNA uptake by specific immune cell types in the spleen (Figure 3D). Significant correlations were observed between the T cell response and early mRNA association with splenic monocytes, macrophages, plasmacytoid dendritic cells (pDCs), and B cells (Figure 3E). In contrast, such correlations could not be established between T cell responses and mRNA association with non-parenchymal liver cells (Figures S4C and S4D).Figure 3LNP immunogenicity correlates with splenic immune cell associationShow full caption(A) A mixture of 5 μg Cy5-labeled (25% substituted Cy5-UTP, 75% substituted 5-methoxy UTP) and 5 μg unlabeled (and without nucleoside modifications) Fluc mRNA in LNPs was injected i.v. (n = 2–4). After 4 h, kidneys, lungs, heart, liver, and spleen were isolated. A part of spleens and livers was kept for immediate flow cytometry analysis to assess Cy5 association with multiple cell types. Remaining tissues were homogenized and assayed for luciferase activity. (B) Luciferase activity per mg of tissue. (C) Luciferase activity (expressed as percent of luciferase activity per mg in all assayed organs) was mainly localized in liver and spleen. (D) Association of Cy5-mRNA with immune cell types in the spleen, expressed as difference in Cy5 mean fluorescence intensity between cells of LNP-injected mice and vehicle-injected mice (ΔCy5 MFI). (E) Correlations of ΔCy5 MFI after first LNP administration with T cell response after three immunizations (Figure 1C) were strongest for monocytes, pDCs, macrophages, and B cells based on Pearson's correlation coefficients. In (B), (C), and (D), mean ± SD is shown. RLU, relative light unit.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Optimal LNP compositions increase mRNA uptake by splenic immune cells and trigger enhanced innate responsesTo further validate these findings, we compared the organ-specific and splenic cell distribution of the o